Automated microscopic cell analysis

ABSTRACT

This disclosure describes single-use test cartridges, cell analyzer apparatus, and methods for automatically performing microscopic cell analysis tasks, such as counting and analyzing blood cells in biological samples. A small measured quantity of a biological sample, such as whole blood, is placed in a mixing bowl on the disposable test cartridge after being inserted into the cell analyzer. The analyzer also deposits a known amount of diluent/stain in the mixing bowl and mixes it with the blood. The analyzer takes a measured amount of the mixture and dispenses in a sample cup on the cartridge in fluid communication with an imaging chamber. The geometry of the imaging chamber is chosen to maintain the uniformity of the mixture, and to prevent cells from crowding or clumping as it is transferred into the imaging chamber by the analyzer. Images of all of the cellular components within the imaging chamber are counted and analyzed to obtain a complete blood count.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/803,897, filed Feb. 27, 2020 which is a continuation of U.S.application Ser. No. 15/616,327, filed Jun. 7, 2017, which claimspriority to U.S. provisional application No. 62/394,702, filed Sep. 14,2016, and to U.S. provisional application No. 62/360,236, filed Jul. 8,2016, and which is a continuation-in-part of U.S. application Ser. No.15/221,285, filed Jul. 27, 2016, which is a continuation of U.S.application Ser. No. 15/017,498, filed Feb. 5, 2016, which is acontinuation-in-part of U.S. application Ser. No. 14/947,971, filed Nov.20, 2015, which claims priority to US provisional applications no.62/138,359, filed Mar. 25, 2015, 62/113,360 filed Feb. 6, 2015, and62/084,760, filed Nov. 26, 2014. All of the above-listed applicationsare herein incorporated by reference.

FIELD OF THE INVENTION

This invention pertains to analyzers and methods for automaticallyperforming microscopic cell analysis tasks, such as counting blood cellsin biological samples. More specifically, the present disclosure relatesto single use devices, apparatus and methods used to count red bloodcells, white blood cells and platelets, and measurements related tothese particles.

BACKGROUND OF THE INVENTION

There are a variety of methods for enumerating particles, such as bloodcells, in a biological sample. Determining the number of cells per unitvolume in a sample provides the physician important diagnosticinformation. The most elementary method of counting cells consists ofintroducing a diluted biological sample into a hemocytometer andexamining it with a microscope. A hemocytometer is a device with anoptically clear chamber having a known depth, typically 100 microns, andruled markings to define a unit volume, typically 0.01 μL. A uniformmixture of diluted whole blood, for example, may be introduced into thehemocytometer by capillary action to form a monolayer. Using amicroscope to visualize the diluted sample, cells of different types canbe counted manually in a limited number of marked areas. Counts areaggregated to compute the number of cells per unit volume. This manualmethod is time consuming, tedious, and requires a skilled technician tooperate the microscope and to recognize the various types of cells, andis prone to error. Its accuracy is limited by the number of cellscounted and the uniformity of the monolayer formed by introduction ofthe diluted sample.

Consequently, automated methods, such as impedancemetry (Coulterprinciple U.S. Pat. No. 2,656,508) and flow cytometry, have beendeveloped for rapid counting, sizing, and classification of a relativelylarge number of cells for diagnostic tests such as the Complete BloodCount (CBC), sometimes referred to a CBC with a five part differential.These automated methods also have shortcomings. The analyzers arerelatively large and expensive and require skilled operators for theiruse and maintenance. Such analyzers are typically available only incentralized laboratories. Blood samples are collected in specialcontainers having an anticoagulant to keep the blood from clotting whilebeing transported to the lab. This process adds costs and risk oferroneous results from transport, handling, labeling and transcription,as well as a time delay in obtaining the results. These analyzers alsoflag or reject in excess of 20% of the tested samples for further reviewby a manual differential. Only highly skilled technicians can perform amanual differential. A flag is commonly generated by impedance or flowcytometry counters because the impedance or scatter profiles of apopulation of cells are ambiguous. Microscopic imaging analysis is notsusceptible to the same ambiguities as impedancemetry and flowcytometry, and thus is used as a reference method. Similarly, automatedimaging analysis have a much lower flag rate.

The CBC generally includes measures of white blood cells (leukocytes)per unit volume (WBC), red blood cells (erythrocytes) per unit volume(RBC), platelets (thrombocytes) per unit volume (PLT), hematocrit (HCT)or packed cell volume (PCV), hemoglobin (HGB), and measurements relatedto red cells including mean corpuscular volume (MCV), mean corpuscularhemoglobin (MCH), mean corpuscular hemoglobin content (MCHC), and redcell distribution width (RDW). A diagnostic test sometimes referred toas a “CBC with differential”, or “CBC with five part diff”, will alsoinclude neutrophil granulocytes (NEU), Lymphocytes (LYM), Monocytes(MON), Eosinophil granulocytes (EO) and Basophil granulocytes (BASO) perunit volume or as a percentage of the white blood cells (WBC). The CBCwith differential also may include counts of Immature Cells (IC),atypical lymphocytes, nucleated Red Blood Cells (nRBC), andReticulocytes (RETIC) per unit volume of the blood sample.

The CBC provides a panel of blood cell measurements that can be used todiagnose a wide variety of abnormal conditions, such as anemia orinfection, or to monitor a patient's treatment, such as chemotherapy.Because of its usefulness, the CBC analysis is one of the most commonlyperformed diagnostic tests in medicine, but patients typically wait aday or more for results. If microscopic cell analysis could be performedin a portable, easy-to-use, analyzer close to the patient, results couldhave a more immediate impact in improving patient care. A simple systemable to provide the CBC in the physician's office, or at bedside, or inthe Critical Care Unit (CCU) or Intensive Care Unit (ICU), or in thehospital emergency room within a few minutes and using a drop of bloodfrom a finger-stick, could have enormous impact on the delivery andaffordability of health care.

Recent patent documents have described simpler devices than centralizedlab hematology analyzers for performing cell analysis of blood samples.In U.S. Pat. No. 7,771,658, issued to Larsen, the applicant, providestechnology for performing a flow-cell analysis of blood cells in asingle use disposable cartridge. Larsen describes means for taking anexact amount of blood sample, diluting the amount of blood with aprecise volume of diluent, and mixing the blood with the diluent toobtain a homogeneous solution. Larsen utilizes a single use cartridge toflow a measured amount of the mixture of sample and diluent through anorifice at a rate of several thousand particles per second, and counts,sizes, and classifies the particles for analysis in accordance with theCoulter principle. Because Larsen's disclosure is directed to theanalysis of a small sample of whole blood, errors in metering variousvolumes or in the mixing or sampling steps can significantly impact theaccuracy of the results.

PCT Patent Number WO 2014099629 issued to Ozcan et al. describes asystem for analyzing a blood sample with a mobile electronic devicehaving a camera. The sample preparation process for each test requiresaccurate measurement of 10 μL, of whole blood to be mixed with 85 μL,phosphate buffered saline and 5 μL of nucleic acid stain. Ten (10) μL ofthis diluted mixed sample are then loaded into a cell counting chamberwith precise channel height of 100 μm and are imaged by a digitalcamera. A separate cell counting chamber is needed for analysis of redcells, white cells, and hemoglobin, and each test should be performedseparately. Accuracy of the final result is not only dependent on theaccurate measurements of the various sample preparation steps, includingthe precise metering of the sample and diluent, but also on the precisefabrication of the counting chambers. Maintaining uniformity andconsistency of the 100 μm dimension of the channel height in adisposable cartridge is difficult to achieve in a low cost device.

U.S. Pat. No. 8,837,803 to Wang et al. describes a method fordetermining cell volume of red blood cells, which seeks to avoid theerrors associated with diluting, mixing, and sampling, by analyzing asample of substantially undiluted whole blood. In theory this approachhas appeal, but the handling of undiluted whole blood is challenging.The cells are so numerous (for example 5,000,000 red cells in 1 thattheir distribution can be impacted by contact with the surfaces of adisposable cartridge. Additionally, in order to image cells in thisovercrowded environment, the imaging chamber should have a depth of onlya few microns to prevent the overlapping of cells, and it should befabricated accurately, because it determines the volume of the bloodsample being analyzed.

Therefore a compact, accurate method of performing microscopic cellanalysis using digital camera imaging of a diluted sample, which doesnot require accurate measurement of the diluent volume, or requireaccurate or precise dimensions of an imaging chamber, is highlydesirable.

SUMMARY OF THE INVENTION

An objective of the present invention is to provide an apparatus thatcan provide counts of cells or other particles of a biological sampleper unit volume without requiring an operator having specializedknowledge or specialized skills. Another objective of the presentinvention is to provide an easy-to-use analyzer that can perform all ofthe measurements of the CBC in a few minutes. Another object of theinvention is to provide an analyzer to perform a CBC on a small fingerstick sample. Another objective of the present invention is to provide amethod for determining the concentration of particles in a dilutedbiological sample that does not require accurate measurement of a volumeof diluent or reagent mixed with the sample. Another objective of thepresent invention is to provide ready-to-use reagents and diluent foruse in performing the microscopic analysis of a biological sample thatdo not require preparation, mixing or measurement by the user. Anotherobject of the invention is to create a substantially homogenousmonolayer of cells of a diluted sample in a single use disposable deviceand to perform a CBC analysis in a few minutes. Another objective is toprovide a sample collection device that can easily be operated by a userto obtain a biological sample for analysis. Yet another objective is toprovide all of the fluidic components of a CBC analyzer in a single-usedevice, so as to prevent cross contamination between samples and toavoid the need for cleaning and washing of fluidic components of ananalyzer. Another objective of the present invention is to provideinternal monitoring of the critical process steps of counting cells, sothat a potentially erroneous result can be flagged. Still anotherobjective is to provide a method of performing the CBC on a sampleimmediately after collecting it, and to eliminate transport of thesample to a laboratory or storage of the sample. Some or all of theseand/or other objectives or advantages may be accomplished by embodimentsof the invention as provided for in the appended claims.

A further object of the invention is to provide a hematology analyzerthat performs a CBC using imaging technology instead of the Coulterprinciple of flow cytometry. Another object of the invention is toprovide a hematology analyzer that utilizes a single use disposableimaging chamber. Another object of the invention is to provide asubstantially homogenous monolayer of cells and platelets of dilutedwhole blood in their unrestrained state incontradistiction to cellssmeared or sprayed on a glass slide, wherein the cells are mechanicallydeformed or distorted or flattened, or cells that are sprayed ordeposited on a glass slide and fixed. Another object of the invention isto provide an imaging based hematology analyzer that flags samples at alower rate than hematology analyzers that use impedancemetry or flowcytometry.

The disclosure provides improved methods, systems, and devices forperforming counts and measurements of particles in a biological sample.Aspects of the disclosure are directed to determining the concentrationof one or more types of particles of a biological sample and providingthe result as the number of such particles per unit volume in a fewminutes. The particles may be any mass suspended in a liquid that can berecognized by optical inspection using an automated microscope and imageanalysis techniques well known in the art. Examples of particlesinclude, but are not limited to, blood cells, platelets, sperm,bacteria, spores, and inorganic particles.

While embodiments of the present invention can be used in manyapplications to count particles suspended in a liquid, the presentdisclosure will demonstrate benefits of the invention in performing theComplete Blood Count (CBC) or CBC with differential on human or animalblood samples as defined above. The present disclosure describes asingle-use test cartridge for use with an apparatus that includes anautomated microscope for analyzing cells in a biological sample. Thetest cartridge is used to collect a biological sample. For example theuser can prick the finger of a patient and obtain a whole blood sampleand collect the resulting drop of blood in the test cartridge. The usercan accomplish this by holding the test cartridge beneath the hangingdrop from the patient's finger and then bringing it closer until thedrop contacts an input port or sample cup on the test cartridge. In analternate embodiment, the user may take a blood sample intravenously andtransfer it to the test cartridge using a plastic bulb pipette. In oneexample the single-use test cartridge includes an input port adapted toaccept a variety of sources of biological samples including a directsample, capillary tube, or transfer pipette. In another example, thetest cartridge could draw a sample into it by capillary action. The testcartridge may also include a closure that the user applies to cover theinput port after the sample has been collected into the test cartridge.The closure may be part of the test cartridge or a separate device. Theuser may apply the closure manually or the apparatus may automaticallymove the closure to cover the input port when the test cartridge isdocked to the apparatus. The closure provides a physical barrier toavoid subsequent contact with any excess sample on the surface of thetest cartridge. The closure has a vent to provide an air path to theinput port to allow the liquid sample to move within the test cartridgeif a vacuum is applied, as will be described below.

Within the test cartridge, the input port is connected by an inputchannel to a metering chamber capable of precisely separating a smallvolume of the sample from the unmeasured sample. As an example, thecollected sample volume may be 5-20 μL, of which the metering chamberseparates out or isolates 0.1-5 μL for measurement. If a finger sticksample is utilized, a sample volume less than 10 μL, will minimize therisk of hemolysis and the risk of dilution by interstitial fluids. Themetering chamber can be a section of a fluid channel, a cavity, or apass-through conduit within a valve, or another volumetric shape thatcan be reproducibly fabricated to contain a predetermined volume of thebiological sample in the range of 0.1 to 5 μL. Injection molding,compression molding, etching or other processes known to those skilledin the making of lab-on-a-chip or microfluidic devices can be utilizedto manufacture the metering chamber.

In one example, the metering chamber is combined with a rotary valvestructure by molding a pass-through conduit in the cylindrical stem ofthe rotary valve. As an example a pass-through conduit having acylindrical cross section with 0.5 mm diameter and a length of 5 mm, hasan internal volume of approximately 1 μL. The pass-through conduit isinitially filled with the biological sample by providing a fluidcommunication path between the input port and input channel, thepass-through conduit, and a vacuum channel. A vacuum applied to thevacuum channel pulls the sample into the pass-through conduit.Alternatively the biological sample can be pulled into the pass-throughconduit by capillary action. Once the biological sample has completelyfilled the pass-through conduit, the cylindrical stem of the rotaryvalve is rotated until the pass-through conduit is no longer connectedto the input channel or the vacuum channel, thus separating or isolatingthe sample volume contained in the pass-through conduit. Alternatively,a rotary face seal valve, a slide valve, or a fixed volume in the testcartridge could be used to isolate a small volume of the sample.

Further, elements of the test cartridge include a prepackaged liquidreagent or alternatively, a chamber for storing a liquid reagent,together with a mixing chamber, an imaging chamber, and fluid channelsthrough which the sample and liquid reagent may be moved. In oneexample, after the biological sample has filled the pass-throughconduit, the rotary valve is positioned so that the pass-through conduitis placed in fluid communication with both a liquid reagent and themixing chamber. Empirical studies have determined that a volume ofliquid reagent or diluent or stain (referred to hereafter as“diluent/reagent”) that is in excess of 3-times the volume of thepass-through conduit is sufficient to wash out the entire isolatedsample. In one example, the diluent/reagent is a combination of diluentand stain and is supplied in a volume to provide a dilution ratio ofabout 40:1. Therefore forty times the volume of the metering chamber canbe used to push the isolated sample out of the pass-through conduit andinto the mixing chamber, where the sample is uniformly mixed with thediluent/reagent, and then transferred into the imaging chamber.

The single use test cartridge contains an imaging chamber, through whichan automated microscope can acquire images of cells in the mixture ofdiluent/reagent and sample. In one embodiment of the present invention,the isolated sample volume is diluted with a known volume ofdiluent/reagent, whereby the dilution ratio is established. The volumeof the diluent/reagent may be determined by measuring it, for example,by monitoring its flow in a fluid channel of known dimensions with acamera or other fluid sensor such as optical reflective/transmitted orultrasonic, or by temporarily metering it into a known volume chamberand then using only that known volume for sample dilution. According tothis embodiment, the cells in a known volume of the diluted sample arecounted, and because the dilution ratio is known, the cell counts perunit volume of the blood sample can be determined. The volume of thediluted sample can be determined by filling an imaging chamber of knownvolume with the diluted sample. The volume of the imaging chamber may beknown by using highly reproducible manufacturing processes, or bymeasuring each test cartridge at time of manufacture and encoding sizingparameters in the package labeling. Alternatively, a measured amount ofthe diluted sample can be transferred into an imaging chamber of unknownvolume and all the cells in the chamber are counted. A measured amountof the diluted sample can be determined by monitoring its flow from themixing chamber into a fluid channel of known dimensions by a camera, andisolating a segment. The segment, in turn, is moved into the imagingchamber.

In a preferred embodiment, the isolated sample volume is transferred tothe mixing chamber and then to the imaging chamber. According to thisembodiment, neither the dilution ratio nor the volume of the diluentneeds to be known. The size of the imaging chamber is chosen to ensurethat the entire volume of the isolated sample and the diluent/reagentcan be contained within the imaging chamber, but the exact dimensions orvolume of the chamber do not need to be known. The depth of the imagingchamber should be small enough to prevent the cells from overlapping atthe chosen dilution ratio when the cells settle to the bottom. Thisdepth is preferably between about 10 μm and 200 μm. In one embodiment,the depth of the imaging chamber is 100 μm and the ratio of thediluent/reagent to the isolated sample is 40 to 1. The width of theimaging chamber is chosen to provide uniform filling by the differentcell sizes and smooth flow without forming voids or crowding of cells.The length of the imaging chamber is calculated based on the depth andwidth parameters to provide the volume needed to accommodate the entireisolated sample at the chosen dilution ratio, and to provide a safetymargin. The shape of the imaging chamber may further be chosen to matchthe field of view of the digital camera or to facilitate capture ofmultiple images or to maintain a uniform distribution of cells

We have found that if the shape of the imaging chamber is square orrectangular having a ratio of length to width of 2:1, the cells will notuniformly fill the imaging chamber. If the mixture of sample anddiluent/reagent is uniform when it enters the imaging chamber, the cellswill tend to bunch and crowd, particularly near the sides or edges, andwhen they settle, the layer on the bottom of the imaging chamber willnot be homogenous. It is important to obtain a substantially homogenousmonolayer of cells in the imaging chamber, as this facilitates thecounting of all the cells. We have found that if the width and depth ofthe imaging chamber are small compared to the length, the cells remainsubstantially uniformly distributed. Desirably, the length-to-widthratio of the imaging chamber is greater than 10:1. In variousembodiments of the present invention, the shape of an imaging chambermay be serpentine, helical, or castellated according to the form factorof the test cartridge. In all of these cases the width and depth aresmall compared to the length so that the cells due not aggregate on thesides or corners of the imaging chamber, and the layer of cells settlingto the bottom of the imaging chamber is substantially homogenous. Thoseskilled in the art will recognize that other geometries for the imagingchamber, which maintain the distribution of cells when the mixedsolution of sample and diluent/reagent is transferred into the imagingchamber may also be utilized.

The design goal of the imaging chamber is to contain all the cells fromthe original metered chamber and the diluent/reagent in a uniform mannerand without significant cell overlap when the cells settle to the bottomof the imaging chamber. The dilution ratio combined with the depth ofthe imaging chamber can be chosen to minimize the overlapping. As anillustrative example, an imaging chamber may have a width between 0.5 mmand 2.5 mm, a depth from 10 to 200 μm, and the dilution ratio may befrom 10:1 to 100:1.

Materials, which contact the cells outside of the imaging chamber, maybe chosen to have surface properties to minimize cell adherence. Liquidreagents, which may include a surfactant or cell sphering agent tofacilitate cell analysis, may also advantageously minimize red cellsbeing lost during transfer or being overlapped in the imaging chamber.The volume of liquid reagent and the flow velocity may also be chosen toimprove the likelihood of transferring every cell from the metering ormixing chamber to the imaging chamber and insuring that the distributionof the cells remains uniform. Yeh-Chan Ahn describes the complexconvective diffusion phenomena that is created in a serpentinemicrochannel which has a varying curvature (See Investigation of laminardispersion with optical coherence tomography and optical Dopplertomography, Yeh-Chan Ahn, Woonggyu Jung, Jun Zhang, and Zhongping Chen,OPTICS EXPRESS 8164, Vol. 13, No. 20, 3 Oct. 2005). Particles, or in ourcase, cells which are in suspension and moving through such amicrochannel tend to segregate according to their size, density, channelshape and flow velocity. Secondary flow from the serpentine geometry cancreate vortices as the channel curves causing the cells to be re-mixedinto the center of the flow at certain fill velocities. This re-mixinghelps maintain a near uniform distribution of the cells as the fluidfills the microchannel. In one embodiment, we have found that aserpentine path that is 1.25 mm wide with turning inside diameter of1.25 mm and an outside diameter of 2.5 mm, a depth of 0.125 mm, a lengthof 500 mm, and an effective fill speed of about 24 μL/s insures that theflow of cells remains uniform.

It should be noted that the time to count every cell is directly relatedto the volume of the metered chamber, and that this creates a trade-offin overall performance. Manufacturing highly reproducible meteringchambers is challenging for very small volumes. However, while it may beeasier and more reproducible to manufacture a metering chamber with a 5μL volume than one with a 1 μL volume, it would potentially take fivetimes longer to count every cell, and analyze them, in a 5 uL volume ofsample than a 1 uL sample volume. Embodiments of the present inventioncan provide a solution to this dilemma. By ensuring that the sample anddiluent/reagent are well mixed and that a proper dilution ratio ischosen, and by utilizing a serpentine shaped imaging chamber where thelength is large compared to the width and depth, the pattern of cellsacross the imaging chamber is relatively uniform and reproducible. Underthese conditions, the layer of cells settling to the bottom of theimaging chamber will be a substantially homogenous monolayer. As aresult, instead of imaging the entire layer and counting every cell, onecan take representative images or frames that are statistically derivedto accurately account for every cell. Thus, within the allowable errorfor the final result(s), every cell is included in the analysis, whetherby actual image or by statistical representation. As an illustrativeexample, the camera may take up to 20,000 images at 20× in order to scanthe entire imaging chamber. Alternatively, every tenth frame could betaken to obtain a statistical representation. Another option would be todivide the imaging chamber into segments and count the cells in everyother segment or every third segment and so on. Another alternative todecrease the imaging time would be to scan the entire imaging chamber at10×/0.25 numerical aperture (NA) or 4×/0.1NA to obtain the total WBC andRBC total counts, and then take images at higher power (20×0.04NA orhigher) to obtain the higher-resolution detail needed for countingplatelets, reticulocytes, and performing the WBC differential.

Embodiments of the present invention can achieve accurate resultsindependent of the many factors that have impacted the accuracy ofresults in the prior art. For example, the volume of the diluent/reagentand the dilution ratio do not impact the results, so long as all thecells from the metered sample volume are transferred to the imagingchamber and are not overlapping or crowded, and presented for analysis.Similarly the representativeness of a selected portion of the mixture ofsample and reagent, and the homogeneity of the portion, which directlyaffect the accuracy of results by prior art methods, need have no impacton embodiments of the present invention. Most importantly, the depth anduniformity of the imaging chamber, which has been difficult or expensiveto control in other prior art efforts, need not impact the accuracy ofresults according to embodiments of the present invention.

The present disclosure further describes a cell analyzer that is smalland easy to use. The cell analyzer accepts the test cartridge andcarries out all the steps of the cell analysis without further inputfrom the user. In one embodiment, the test cartridge contains alldiluents and reagents needed to perform a CBC analysis of the patientsample placed in it. In this embodiment, the cell analyzer has fluidhandling components including positive and negative pressure sourcesthat interface with the test cartridge when it is placed into theanalyzer. One or more connections are made between the analyzer and thecartridge to place these pressure sources into fluid communication withchannels in the cartridge to provide a motive force to move liquidswithin the cartridge. The cell analyzer also has a mechanical valvedriver for operating a valve in the test cartridge for controlling themovements of fluids in the test cartridge. When the test cartridge isplaced in the analyzer, the mechanical valve driver is connected to avalve indexer to provide means for operating the valve in the testcartridge. The cell analyzer includes a mechanism for release of thediluent/reagent that are stored on board the test cartridge. The cellanalyzer further includes fluid control logic to automatically controlmovement of the fluids in the test cartridge by activating the positiveand negative pressure or displacement sources and operating themechanical valve driver according to pre-programmed sequences. The cellanalyzer may include a process monitoring camera positioned to acquiredigital images of the movement of fluids in the cartridge. Informationfrom the process monitoring camera can be used to provide feedback forthe fluid control logic or for monitoring critical steps.

The present disclosure also provides devices and methods for managingand/or monitoring the sample collection and preparation process toensure accurate results. In applications where the present invention isused to perform a CBC analysis, if too much time lapses betweenobtaining the blood sample and completing sample dilution and stainingwithin the test cartridge, the blood may clot or the cells may settleresulting in erroneous results. In one embodiment of the presentinvention that mitigates this risk, the test cartridge is placed on theanalyzer in a slide-out tray which initiates process monitoring withinthe analyzer. The blood sample is added to the cartridge by dispensing afree-hanging drop, or by using a capillary tube that is inserted intothe test cartridge or by transferring a sample and depositing it with apipette. Immediately after the sample has been added to the cartridge,the user presses “Run Sample” to initiate the test sequence. Because thecartridge is controlled by the analyzer, the time to collect the sampleis known and can be short enough to avoid the need for anticoagulantcoating or mixing the blood sample.

In an alternate embodiment that allows the sample to be collectedseveral minutes before processing, an anticoagulant, such as K2 or K3EDTA, is provided. This may be achieved by coating the sample input cupwith anticoagulant, by use of a capillary tube coated with anticoagulantfor a finger-stick sample, or by sampling from an evacuated bloodcollection tube such as a Becton Dickenson Vacutainer® containinganticoagulant. To manage cell settling, the test cartridge according tothis embodiment has a timing indicator which is initiated when the inputport is opened or the blood sample is introduced. The timing indicatorcan be a color-changing chemical reaction, a time delayed thermalreaction, an analog or digital timer, or other means known in the art.When the user loads the test cartridge into the analyzer, the timingindicator is read and if needed, the sample is mixed before proceeding.If too much time has lapsed the sample can be rejected to avoidproducing erroneous results.

In yet another embodiment that facilitates remote sample collection, thetest cartridge is docked to a carrier system. The carrier system is ahandheld or portable device used to facilitate a portion, or all, of thesample preparation steps. After blood is added to the test cartridgeusing any of the above mentioned methods, the carrier system accordingto this embodiment draws the blood into the cartridge, meters thesample, performs quality checks, and completes the sample preparation topresent the cells for image analysis. The carrier system would theneither eject the test cartridge for the user to transfer to the imaginganalyzer, or the carrier system could be docked to the imaging analyzerfor an automated handoff. In this embodiment anticoagulant coatings arenot needed and there is no risk of cell settling because the criticalsteps are initiated as soon as the sample is placed in the testcartridge.

Combinations of the workflow devices and methods are also contemplatedin the present disclosure. By way of example, a simple carrier systemcould incorporate a digital timer and a mechanism able to meter thesample, but not perform quality checks or full sample preparation. Theseadditional steps would be done by the cell analyzer.

The cell analyzer contains an automated microscope including anobjective lens, focusing mechanism, bright-field and/or fluorescentlight sources or both, filters, a dichroic mirror and a digital camera.In some embodiments, the cell analyzer may further include anillumination source and photometric detector for measuring lighttransmission at one or multiple wavelengths for measuring theconcentration of an analyte in the sample. For example, the testcartridge can include a photometric chamber which is in fluidcommunication with the input port by means of a fluidic channel, andthrough which the sample can be transferred for photometric analysis,such as a hemoglobin measurement.

By way of example, to measure hemoglobin, the cell analyzer may have anillumination source consisting of two light emitting diodes (LEDs)providing excitation at wavelengths of 502 nm and 880 nm. The light pathfor the LEDs and a photometric detector are approximately 0.030″diameter. The 502 nm LED may be selected for absorbance measurementbecause of an isobestic point of oxyhemoglobin (O2Hb) anddeoxyhemoglobin (RHb). Also at 502 nm, the slope of the O2Hb and RHbcurves are very low, resulting in minimal variation with varying oxygensaturation (sO2) levels. Furthermore, the carboxyhemoglobin (COHb) curveis also close to this isobestic point, resulting in very little effectfrom COHb. An 880 nm LED can measure the background scatter effectscaused by RBCs, WBCs, lipids, etc. This is particularly important whentaking measurements on whole blood. This measurement may also beperformed on lysed blood, with or without a reagent for converting thehemoglobin to a single form, such as reduced hemoglobin, methemoglobin,azidemethemoglobin or cyanomethemogloin. In the case of a conversion toa single form of hemoglobin, a different peak wavelength for absorptionmeasurement may be used, such as 540 nm or 555 nm.

Disclosed and contemplated aspects also include an example of a testcartridge that is not preloaded with reagents, and instead, is coupledto a reagent supply module contained on board the cell analyzer. Theuser loads the reagent supply module into the cell analyzer where it isutilized for multiple test cartridges. When the reagent supply module isexhausted or expires, the cell analyzer alerts the user and will notperform additional tests until the reagent supply module is exchanged.The reagent supply module includes a cradle for receiving the testcartridge, a vessel for holding a liquid diluent/reagent, a diluentdelivery pump in fluid communication with the vessel, and adiluent/reagent output port constructed to interface with the testcartridge when the cartridge is in the cradle. In one example the sizeof the vessel is of sufficient capacity to provide diluent/reagents todilute several samples (50-100) with a reagent to sample ratio of 10:1to about 250:1. The diluent/reagent supply module may include aself-priming mechanism for priming the liquid reagent and eliminatingair bubbles. ‘The reagent supply module may further include a chamberfor collecting waste diluent/reagent from the priming process.

In another embodiment contemplated in the present disclosure, a smallmeasured volume of whole blood may be mixed manually with an impreciseamount of diluent/reagent in a sample preparation device. For instance,the known volume of sample and imprecise diluent/reagent may be put intoa sample tube and gently rocked back and forth a few times. The entiremixed volume is then transferred by using a transfer pipette or similardevice to a test cartridge having an imaging chamber sufficiently largeto contain the entire mixed volume. According to embodiments of thepresent invention, if all of the cells in the imaging chamber arecounted (either directly or by statistical sampling), then theconcentration of cells per unit volume can be determined, without oneneeding to know the volume of the diluent/reagent, the dilution ratio,or the volume of the mixed sample in the imaging chamber, or the volumeof the imaging chamber occupied by the mixture. One drawback to thisembodiment is the practical difficulty in measuring a small volume ofsample, e.g. 1 uL. If a large volume of sample is chosen, such as 10 uL,it will take a longer time to count all the cells and it will require arelatively large imaging chamber. In the case of a 10 uL sample size anda dilution ratio of 50:1, an imaging chamber of 500 uL would berequired. An alternative embodiment would be to take a 10 uL sample andmix it with a precise volume of diluent, and then taking a portion ofthe mixture and transferring it into an imaging chamber sufficientlylarge to contain the portion of mixture. If every cell of the mixture iscounted, the number of cells per unit volume can be easily determined,since the dilution ratio is known.

Diluent/reagents that are preloaded in the test cartridge, or areprovided in a separate sample preparation device, or by a supply moduleare in a ready-to-use format. For a CBC analysis, the reagents mayinclude a membrane-permeable dye, such as Acridine Orange todifferentially stain the DNA and RNA of cells in whole blood. Otherstains known to those skilled in the art, such as cyanine dyes, can alsobe used to stain the blood cells. In an alternative arrangement, thestain may be provided in a dry reagent form together with a diluent thatis mixed with the dry reagent as needed. Multiple stains can be includedin a combination reagent. In one embodiment, the reagents may include anantibody conjugated to a detectable label that targets specific cells orspecific antigens associated with cells. The detectable label may be adye, a fluorescent dye, quantum dot, colloidal metal such as gold,silver, or platinum or other detectable constructs known in the art. Thedetectable label can also be used to detect a cell-specific antibody,such as CD3, CD4, CD14, CD16, CD19, CD34, CD45, CD56, or any other ofthe enumerated Cluster of Differentiation markers. The detectable labelcan also be used to detect bacterial or parasitic pathogens, platelets,recirculating tumor cells, leukemic cells, stem cells or any combinationof these.

In other embodiments the liquid reagent may contain a surfactant such aspolysorbate or sodium dodecyl sulfate (SDS), an anticoagulant such asEDTA, and/or a sphering agent such a zwitterionic detergent to provideisovolumetric reshaping of the red blood cells to facilitate cell sizemeasurement and computation of the mean corpuscular volume (MCV).

Systems according to the invention can exhibit better quantitativeaccuracy than manual microscope analyses using a manual hemocytomer orsimilar device, which tend to be limited by variability in samplepreparation and limited counting statistics. In embodiments of thepresent invention, sample preparation is improved by removing criticaloperator fluid handling steps and by automation of all dilution steps.Because every cell and platelet is counted in the entire metered volumeof sample, any error in the sample dilution is irrelevant.

Systems according to the invention can also save time that wouldotherwise be allocated to manual hemocytometer slide preparation, setuptime, and microscope focusing, which can limit the number of bloodsamples that can be analyzed. Automation can greatly increase the rateof image acquisition and analysis, allowing for more cells to beanalyzed and counted. This can improve the counting statistics andoverall precision of the system.

Systems according to the invention can also extend the capabilities ofcell counting methods by enabling CBC point-of-care testing, i.e. nearpatient testing, to permit immediate clinical decisions to be made.Personnel having a relatively low skill level can operate the systems.The analyzers can be engineered to be inexpensively manufactured andeasily serviced, allowing them to be more readily deployed atpoint-of-care sites, such as at the patient's bedside, in physician'soffices, and at emergency sites.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

In the drawings, like reference characters generally refer to the sameparts throughout the different views. The drawings are not necessarilyto scale, emphasis instead being placed upon illustrating the principlesof the invention. In the following description, various embodiments ofthe present invention are described with reference to the followingdrawings, in which:

FIG. 1 is a perspective view of an illustrative test cartridge beingpositioned to collect a drop of blood from a patient's finger;

FIG. 2A is a perspective view of an illustrative test cartridge with acover shown in the open position to receive a biological sample;

FIG. 2B is a perspective view of the test cartridge shown in FIG. 2Awith the cover shown in the closed position ready for analysis;

FIG. 3 is a cut-away view of an illustrative cell analyzer showinginternal components with a test cartridge being inserted;

FIG. 4 is a plan view of an illustrative test cartridge of the type thatincludes reagents for conducting a test;

FIG. 5 is a plan view of an illustrative test cartridge of the type thatdoes not include reagents;

FIG. 6 is a perspective view of an illustrative reagent supply moduleshowing a test cartridge ready to be joined with the module;

FIG. 7A is a perspective bottom view of a metering chamber formed in theface of a valve stem of a rotary valve;

FIG. 7B is a side view of a valve stem of a rotary valve with apass-through conduit, which serves as a metering chamber, with ametering chamber;

FIG. 8A is a plan view of an illustrative test cartridge showing asample of whole blood deposited in the input port area;

FIG. 8B is a plan view of the test cartridge of FIG. 8A showing initialmovement of the sample and reagent with the rotary valve in the firstopen position;

FIG. 8C is a plan view of the test cartridge of FIG. 8B with the valvein the second open position;

FIG. 8D is a plan view of the test cartridge of FIG. 8C illustrating thesample and the reagent in the imaging chamber;

FIG. 8E is a plan view of the test cartridge of FIG. 8D illustrating thesample and most of the reagent positioned in the mixing chamber;

FIG. 8F is a plan view of the test cartridge of FIG. 8E illustrating allof the sample and the reagent positioned in the imaging chamber and thevalve in a final, closed position;

FIG. 9 is an side elevation view of a cross section of the passivemixing chamber taken through line 9-9′ of FIG. 8E;

FIG. 10 is a plan view of an alternative test cartridge with sampleinput port and imaging chamber;

FIG. 11 is a flowchart illustrating the operation of the cell analyzer;

FIG. 12 is a plan view of a test cartridge with sample cup andserpentine imaging chamber;

FIG. 13 is a plan view of a test cartridge with a serpentine imagingchamber.

FIG. 14 is a plan view of a hematology analyzer using a single testdisposable test cartridge illustrated in FIG. 15 ;

FIG. 15 is a plan view test cartridge showing a mixing bowl, sample cup,and serpentine imaginging chamber; and

FIGS. 16A and 16B show bright-view and fluorescent images of the samecells that were collected according to the present invention

DETAILED DESCRIPTION

FIG. 1 illustrates test cartridge 100 being positioned to collect a dropof blood 120 from a patient's finger. The test cartridge is held beneaththe hanging drop 120, so that it contacts the input port 130 of the testcartridge 100. The input port 130 comprises a recessed area or openingthat may be coated with an anticoagulant and have surface treatment orfeatures such as small columns to increase surface retention to collectand hold the blood sample. In an alternate embodiment, the blood sample120 may be collected intravenously and introduced to input port 130 by atransfer pipette or capillary tube. The transfer pipette or capillarymay contain an anticoagulant coating according to the desired workflow.The volume of blood or other biological sample placed in input port 130is sufficient to visually fill the recessed sample area, but isunmeasured.

FIG. 2A shows test cartridge 100 with closure 135 shown in the openposition to provide access to input port 130. Closure 135 is adapted toslide relative to the test cartridge 100 and may have detent or otherpositioning features that facilitate placing it in different positions.After the biological sample has been collected into input port 130,closure 135 may be moved to the position shown in FIG. 2B to cover theinput port 130. The closure 135 may be moved by the user prior toinserting it into the analyzer as shown in FIG. 3 . Alternatively,closure 135 may be moved by an operation within the cell analyzer.Alternate embodiments of closure 135 may include graphics, identifyinginformation, or instructions to the user. While the closure 135 isillustrated as a sliding component, other means of closing the inputport 130 are contemplated including a cap that hinges upward, a smallsurface cover that swivels away from and returns to cover the input port130, or an adhesive component that sticks to the input port 130 or areasurrounding it. In all cases the closure 135 includes a vent or air pathto the input port to allow the blood sample to move into the testcartridge 100.

FIG. 3 is a cut-away view of an illustrative cell analyzer 200 with testcartridge 100 positioned so that the operator can introduce it into theanalyzer. From the outside of the cell analyzer 200, one can see thehousing 206, a user-interface screen 208, a printer 212, and a cartridgeloading door 217. When the cartridge loading door 217 is opened, thetest cartridge 100 can be placed on a cradle 220 of x-y stage 225,configured to receive test cartridge 100 from the user. The cradle 220provides mechanical alignment of the cartridge to facilitate connectionsthat are made between the analyzer and the cartridge. For example, amechanical presser foot 230 may be placed in contact with a flexiblesurface on the test cartridge to provide mechanical pressure ontopackaged, on-board reagents. Some embodiments of the cell analyzer 200may utilize a reagent supply module 470 as further described withreference to FIG. 6 . Reagent supply module 470 may be installed on x-ystage 225 and has a receiving area 473 (see FIG. 6 ) to providealignment of the test cartridge 402 with the reagent module 470.

A valve driver 235 can be positioned to operate a rotary valve on thetest cartridge. A vacuum/pressure pump 240 supplies negative or positivepressure to a manifold 245, which interfaces with the test cartridge 100when it is placed in the cell analyzer as described below. The cellanalyzer 200 further includes system controller 250 to control movementof the fluids in the test cartridge by activating the vacuum/pressurepump 240, moving the mechanical presser foot 230, or operating the valvedriver 235 according to pre-programmed sequences. Monitoring camera 255,positioned to acquire digital images of the fluids in the cartridge,provides feedback for the system controller 250. Monitoring light source256 may be a ring illuminator that surrounds the lens of the monitoringcamera 255. Information from the monitoring camera 255 is used toprovide feedback for controlling movement of liquids, for positioningthe rotary valve, and for confirming critical steps.

Also shown in FIG. 3 are the components that comprise the automatedmicroscope of the cell analyzer 200. At the base of the analyzer,bright-field light source 260 provides illumination through the testcartridge to the objective lens 265, operatively coupled to focusingmechanism 267. At the top of the analyzer, fluorescent light source 270provides illumination through dichroic mirror 277 to provide fluorescentexcitation of the sample. At the rear of the analyzer, digital camera280 captures images of the test cartridge 100 and transmits them toimage processor/computer 290. In some embodiments, the cell analyzer mayfurther include a photometric light source 293 and photometric detector295 for measuring light transmission at one or multiple wavelengths in achamber in test cartridge 100, such as for measuring hemoglobin, as ismore fully explained below.

FIG. 4 shows an illustrative test cartridge 401 of the type thatincludes liquid reagents stored in a blister pack 417 for conducting atest. The test cartridge 401 has an input port 407 for receiving asample, a passive mixing chamber 405 for mixing the sample withdiluent/reagent, and an imaging chamber 403 for capturing images of thecells in the mixture of sample and diluent/reagent for analysis. In thisembodiment, photometric chamber 409 may be filled with whole blood tomake optical absorbance measurements to determine concentrations ofcertain analytes in the sample, such as hemoglobin. A rotary valve 415provides fluidic connections between various fluidic channels, vents,and ports, including sample driver port 411, vent 423 and mixture driverport 429 as will be described in FIGS. 8A-8F.

FIG. 5 shows an illustrative test cartridge 402 of the type that doesnot include on-board diluent/reagents. Many of the functional componentsare identical to those illustrated with reference to test cartridge 401,but instead of on-board diluent/reagents, test cartridge 402 has areagent input port 460 adapted to be connected to an external source ofdiluent/reagent. Test cartridge 402 may be used in embodiments in whichdiluent/reagents that are needed for an analysis may be too costly topackage individually or may require refrigerated storage. In such anembodiment, diluent/reagent may be provided from a source within cellanalyzer 200 or from a reagent supply module.

FIG. 6 shows an illustrative reagent supply module 470 positioned toreceive test cartridge 402. The reagent supply module 470 includes areceiving area 473 for docking the test cartridge 402, and contains avessel for holding the diluent/reagent, a reagent metering pump adaptedto pump the diluent/reagent, and a reagent output port 475. The reagentoutput port 475 is constructed with a suitable shape and/or elastomericmaterials to insure a liquid-tight connection to reagent input port 460on the test cartridge 402, when the test cartridge is docked to thereagent supply module 470. Reagent supply module 470 has an opening 477suitably sized to allow monitoring camera 255 (FIG. 3 ) to image therotary valve 415. Additionally a window 478 in the reagent supply module470 is constructed to align with the photometric chamber 409 in the testcartridge. Window 478 allows the photometric light source 293 andphotometric detector 295 (FIG. 3 ) to make optical absorbancemeasurements on the fluid within photometric chamber 409.

In one embodiment, the size of the vessel within reagent supply module470 is of sufficient capacity to provide diluent/reagents to diluteand/or stain from ten to about one-hundred samples with adiluent/reagent to sample ratio of 10:1 to about 250:1. The reagentsupply module 470 further can include a self-priming mechanism forpriming the liquid reagent and eliminating air bubbles. In such anembodiment, the reagent supply module 470 may include a chamber forcollecting waste reagent from the priming process. Once the testcartridge 402 is docked with the reagent supply module 470 the combinedpieces perform the same functions as test cartridge 401 except that thereagent supply module 470 replaces the blister pack 417. Inside cellanalyzer 200 the vacuum/pressure pump 240 makes connections throughmanifold 245 to sample driver port 411 and mixture driver port 429. Theinterfaces between the manifold 245 and these ports are constructed witha suitable shape and/or elastomeric material to ensure an airtightconnection so that system controller 250 can control movement of thefluids in the test cartridge (see FIG. 3 ). In such an embodiment thepresser foot 230 is not needed.

The only volume that is measured precisely is the metered volume of theoriginal biological sample. Various means for metering a small volume ofliquid are well known in the art. Two devices that are well suited forlow cost, single use applications according to the present invention areshown in FIG. 7A and FIG. 7B. FIG. 7A shows the face of a cylindricalvalve stem 485 of a rotary face valve. Metering chamber 483 is formed inthe face by highly precise manufacturing processes such as injectionmolding. The chamber 483 is narrow and tubular in shape and centered inthe face of the cylindrical stem 485. A slot 487 in the top of stem 485acts as a valve indexer to indicate the position of the valve stem 485.Also formed in the face of valve stem 485 is an auxiliary connector 421,which has a circular shape. When assembled into the rotary valve 415(FIGS. 4 and 5 ), metering chamber 483 is able to connect between portsin the valve which are 180 degrees apart, while auxiliary connector 421connects between other ports which are 60 degrees apart. As will beexplained with reference to FIG. 8A-8F, system controller 250 is able tocontrol movement of the fluids by rotating valve stem 485 and bypositioning the valve according to the valve indexer 487 according topreprogrammed sequences. Thus in a first position, the metering chamber483 can be connected to the input port 407 (FIG. 4 and FIG. 5 ) andfilled with the biological sample, and then by rotating valve stem 485,the volume contained within metering chamber 483 can be isolated andtransferred for analysis.

FIG. 7B is a side view of a valve stem 485′ with a metering chamberformed as a pass-through conduit 413 in the tapered seat of valve stem485′. Pass-through conduit 413 is able to connect with fluidic channelsin rotary valve 415 which are 180 degrees apart. Also shown in FIG. 7Bis auxiliary fluidic connector 421′, which provides connections toadjacent fluidic channels which are 60 degrees apart.

When assembled in the rotary valve 415 (FIG. 4 and FIG. 5 ) having atapered seat to receive valve stem 485′, pass-through conduit 413 can beconnected to input port 407 (FIG. 4 and FIG. 5 ), filled with thebiological sample, and then by rotating valve stem 485′, the volume ofsample contained within pass-through conduit 413 can be isolated andtransferred for analysis. FIG. 7B also shows auxiliary fluidic connector421′, which provides fluidic connections to adjacent fluidic channels onthe test cartridge according to the position of the valve indexer 487′.It will be appreciated that the rotary face valve of FIG. 7A and thetapered seat valve of FIG. 7B are alternate embodiments for isolatingsample and controlling fluidic paths. Therefore, in the descriptionsthat follow references to metering chamber 483 in a rotary face valvewill be equally applicable to pass-through conduit 413 in a tapered seatvalve.

Now turning our attention to FIGS. 8A through 8F, and with reference toFIG. 3 , a sequence of operations will be illustrated that enable cellanalyzer 200 to perform automated microscopic cell analysis on abiological sample without skilled operator interactions. In FIG. 8A asample is shown deposited into input port 407, which is in fluidcommunication with rotary valve 415. As illustrated in FIG. 8A, the stem485 (FIG. 7A) of rotary valve 415 is in a first position wherein themetering chamber 483 (FIG. 7A) is aligned with the sample input port 407and the sample driver port 411. A vacuum, supplied by the analyzer tosample driver port 411, draws the sample from the input port 407 intothe metering chamber 483 and into the photometric chamber 409. When thephotometric chamber 409 has been filled with sample, the systemcontroller 250 (FIG. 3 ) collects absorbance data from the undilutedsample using the photometric light source 293 (FIG. 3 ) and photometricdetector 295 (FIG. 3 ). As will be understood by those skilled in theart, suitable choice of optical wavelengths and chamber geometry andanalysis of the light passing through the biological sample can be usedto determine concentrations of certain analytes in the sample such ashemoglobin.

By illustration and with reference to FIG. 8B, cartridge 401 is shownwith a diluent/reagent contained in a blister pack 417. When rotaryvalve 415 positioned such that the metering chamber 483 is aligned withthe input port 407 and photometric chamber 409, auxiliary connector 421provides a fluid communication path between the blister pack 417 andvent 423. When pressure is applied to the blister pack 417 by presserfoot 230 (FIG. 3 ), diluent/reagent is released and flushed throughauxiliary connecter 421 thereby priming the channels and removing airbubbles through vent 423.

FIG. 8C shows rotary valve 415 turned counterclockwise 60 degrees to asecond position, which isolates a predetermined amount of sample in themetering chamber 483. In this second position the stem 485 of rotaryvalve 415 is positioned such that the metering chamber 483 is in fluidcommunication with blister pack 417 and the serpentine imaging chamber403.

In FIG. 8D, the rotary valve 415 is shown in the same position as inFIG. 8C but following operation of the presser foot 230 which appliespressure to the blister pack 417. As illustrated by the shaded area, thediluent/reagent from blister pack 417 and the isolated sample 493 fromthe metering chamber 483 are transferred into the imaging chamber 403. Aminimum volume of reagent of three times the volume of the pass-throughconduit 413 is needed to flush the entire sample from the rotary valve415. According to the analysis being conducted, a sufficient volume ofthe reagent is pushed through the rotary valve 415 to completely washout the isolated sample and to achieve the approximate dilution ratiodesired.

In FIG. 8E the rotary valve 415 is shown turned counterclockwise 120degrees from its previous position shown in FIG. 8D to its thirdposition, wherein auxiliary connector 421 is aligned with mixture driverport 429 and imaging chamber 403. Vacuum/pressure pump 240 of cellanalyzer 200 (FIG. 3 ) supplies pressure to mixture driver port 429 andpushes all of the mixture of sample and diluent/reagent from the imagingchamber 403 into passive mixing chamber 405. As the mixture enterspassive mixing chamber 405, air within the chamber is vented throughvent port 433. Once all of the mixture of sample and diluent/reagent hasbeen transferred to the passive mixing chamber 405, vacuum/pressure pump240 applies a controlled vacuum to mixture driver port 429 such that themixture is pulled back into the imaging chamber 403. A preprogrammedsequence of pushing the mixture into the passive mixing chamber 405 andpulling it back into the imaging chamber 403 is repeated to achieve afinal mixture 495 that is free from cell clumping and overlapping afterthe cells settle to the bottom of the imaging chamber 403. In the finalmovement of the mixture 495, it is positioned entirely within theimaging chamber 403 as illustrated in FIG. 8F. We have found that thatin most instances, pushing the sample and diluent/reagent into mixingchamber 405 and pulling it out is sufficient to provide a uniformmixture. Further, the mixture remains substantially uniform when it istransferred into serpentine imaging chamber 403. It should also be notedthat the mixing chamber 405 could be located at the beginning of theimaging chamber 403.

FIG. 8F illustrates the final step of the sample preparation sequence.At this point in the preprogrammed sequence, the entire final mixture495 has been withdrawn from the passive mixing chamber 405 and ispositioned in the imaging chamber 403. When this position is achieved,the rotary valve 415 is rotated counterclockwise approximately 30degrees to the position shown in FIG. 8F, whereby it is not in fluidcommunication with any fluidic channel in rotary valve 415, therebyblocking further fluid communication with the imaging chamber 403 sothat no further movement of the final mixture 495 can take place.

FIG. 9 shows a cross section of the passive mixing chamber 405. Thechamber is referred to as “passive” because as illustrated, it does notcontain any active mixing element such as a bead or spin-bar. Suchdevices may be used in some embodiments, but we have found that anadequately sized chamber as depicted in FIG. 9 is simpler and providesexcellent mixing of the sample and reagent. In operation thediluent/reagent and sample 493 are driven by vacuum/pressure pump 240(FIG. 3 ) and enter and exit the chamber through mixing chamber opening497. As liquid enters the chamber, air within the chamber escapesthrough vent port 433. The cross section of passive mixing chamber 405illustrates wall geometry that increases smoothly in size from thebottom to the top such that the mixture entering from below expands intoa larger volume. The chamber 405 may have asymmetrical sloped walls 484and 491 to promote mixing of the sample and reagent and for removingbubbles from the mixture. After all of the mixture is in the chamber,air bubbles may be introduced to the chamber by vacuum/pressure pump 240through mixing chamber opening 497. These air bubbles further promotemixing and subsequently escape through vent port 433. The choice ofmaterials used to fabricate the passive mixing chamber 405 should takeinto consideration the wetting properties of the specific 1diluent/reagent(s) being utilized in the test cartridge 401. Theproperties of the material, among other requirements, should ensure thatliquid surface tension will pull back all of the liquid in contact withthe side walls of the chamber when the vacuum/pressure pump 240 emptiesthe chamber through mixing chamber opening 497.

FIG. 10 illustrates test cartridge 900 which comprises an imagingchamber 903 having at one end a sample input port 950, and at theopposite end a vent 953. A user of test cartridge 900 collects a smallknown volume of whole blood and mixes it manually with a diluent/reagentin a separate single-use sample preparation device (not shown). Oncemixed, the entire mixed volume is injected into sample input port 950 ata controlled rate, such that the cells uniformly fill the imagingchamber. Air escapes through vent 953, allowing the sample anddiluent/reagent mixture to fill the imaging chamber 903. The form of theimaging chamber is essentially the same as the imaging chamber asdescribed above and shown in FIGS. 8A-8F, except that the volume of theimaging chamber must be sufficient to include all of the mixture ofsample and diluent/reagent. Test cartridge 900 can be placed intoanalyzer 200 (FIG. 3 ) to count every cell in the mixture of sample anddiluent/reagent and for analysis beginning at step 560 of FIG. 11 asdescribed below.

Turning our attention to FIG. 11 we will now describe the overalloperation of cell analyzer 200 configured to provide a “CBC withDifferential” analysis with reference to the test cartridge 401illustrated in FIGS. 8A-8F and cell analyzer 200 illustrated in FIG. 3 .To obtain the blood sample from a patient presented at box 500, the userfirst obtains a new test cartridge 401 at box 505 and opens it to exposethe input port 407. Blood from a finger prick is applied as illustratedin FIG. 1 at box 510 and the input port 407 is covered. The user insertsthe test cartridge into the cell analyzer 200 at box 515. The testcartridge is moved into the analyzer where mechanical and fluidconnections are made between the analyzer and the cartridge as describedabove with reference to FIG. 3 . As a first step of analysis, the sampleis drawn into the metering chamber passing through and into photometricchamber 409 (FIG. 8A). Absorbance of the blood is measured at box 520.Data from absorbance measurements are used to determine hemoglobinconcentration. At box 530 sample in the metering chamber 483 is imagedusing monitoring camera 255 and analyzed to confirm that the meteringchamber was properly filled at box 535. If an error is detected theanalysis is terminated at box 537 and the user is alerted to the errorand instructed to remove the cartridge and reject the test.

If the pass-through conduit 413 is correctly filled the diluent/reagentchannel is primed at box 540 as described above with reference to FIG.8B. Rotary valve 415 is then turned to the position shown in FIG. 8C toisolate the sample and to allow diluent/reagent to wash the meteredvolume of blood out of the pass-through conduit 413 at box 545 whilebeing imaged by monitoring camera 255. The transfer continues until themonitoring camera 255 confirms that diluent/reagent plus sample hasalmost filled the imaging chamber as illustrated in FIG. 8D.

Once a sufficient volume of diluent/reagent is transferred, rotary valve415 is positioned as shown in FIG. 8E and the total volume of sample anddiluent/reagent is mixed at box 550. At box 555 the entire volume 495 istransferred to the imaging chamber and rotary valve 415 is positioned asshown in FIG. 8F. Note that by transferring the entire volume of mixedsample 495, all of the metered volume of blood from the original sampleplus the unmetered volume of diluent/reagent is positioned in theimaging chamber at box 555.

If test cartridge 400 is used, it is inserted into cell analyzer 200 andanalysis begins at step 560. Analysis of test cartridge 401 or 402continues at step 560 when the x-y stage 225 moves the test cartridge401 to obtain bright-field and fluorescent images of the entire imagingchamber 403 at box 560. In an alternate embodiment, objective lens 265and/or digital camera 280 are moved and test cartridge 401 remainsstationary. In yet another embodiment objective lens 265 has sufficientfield of view to capture the entire imaging chamber 403 withoutmovement. Two digital images of each physical frame of the imagingchamber are transferred to image processor/computer 290 at box 565. Oneimage, taken with bright-field optics, can be compared to the otherimage taken with fluorescent optics to identify red blood cells, whiteblood cells and platelets. Further analysis of the white cell sizes andinternal structure can identify sub-types of white cells using patternrecognition.

At box 570 comparison of the bright-field and fluorescent images candifferentiate mature red cells from reticulocytes and nucleated redblood cells. By dividing each cell count by the known volume of themetering chamber 483, the concentration (cells per unit volume) can bedetermined. By using a sphering agent the planar sizes of red cells canbe transformed into mean corpuscular volume (MCV). Combining the redblood cell count with MCV and the volume of the metering chamber 483allows the calculation of hematocrit (HCT) and red cell distributionwidth (RDW). Further calculations using the separately measured HGB frombox 525, combined with the RBC count gives mean corpuscular hemoglobin(MCH), and mean corpuscular hemoglobin content (MCHC).

At box 575 the measured results are compared with previously definedlimits and ranges for the particular patient population anddetermination is made whether the results are within or outside normalexpected ranges. According to this determination results within normalranges are reported in box 580 and results that are outside the normalranges are reported in box 585.

As noted above, another embodiment of the invention is to perform a CBCon a known or measured volume of diluted sample. In this embodiment,every cell and platelet in the known volume of diluted sample iscounted. If the volume of the diluted sample and the dilution ratio isknown, the number of cells and platelets per unit volume of sample canbe determined. A hematology analyzer can be provided to perform the CBCon a known volume of diluted sample, utilizing a single use disposabletest cartridge. The length and depth of the imaging chamber will dependupon the dilution ratio and the volume of the known diluted sample. Forinstance, a 20 uL sample of whole blood may be diluted 50 to 1 producing1000 uL of diluted sample. 20 uL of the diluted sample may then be takento be analyzed. Every cell and platelet in the diluted sample iscounted, either directly or by statistical representation. The 20 uL ofknown diluted volume corresponds to 0.4 uL of whole blood. The volume ofthe imaging chamber must be at least 20 uL in order to contain all ofthe known volume of diluted sample.

The dilution ratio must be sufficient to prevent crowding or overlappingwhen the cells settle to the bottom of the imaging chamber. The dilutionratio also depends on the depth of the imaging chamber as explainedabove. The volume of diluted blood must be sufficient to contain enoughwhite cells to be significantly representative of the whole bloodsample. For example, the average number of white cells in whole blood ofa healthy patient would be approximately 5000 per microliter. In 0.4 uLof whole blood, there would be about 2000 white cells. However, in asick patient, or one being treated with chemotherapy, the white cellcount could be as low as 500 white cells per microliter. In this case,the number of white cells in a 0.4 uL diluted sample would be about 200cells, which may be an inadequate number of white cells to be clinicallysignificant. In this case, a larger volume of diluted sample may bedesirable. However, the time to image and count every cell in the knownvolume of diluted sample increases as the volume of the diluted sampleincreases.

The hematology analyzer utilized to perform a CBC on a known dilutedvolume of sample will comprise an automated microscope for imaging thecells in the imaging chamber of a test cartridge, similar to the onedescribed above and shown in FIG. 3 , except that it need not have apresser foot and valve driver. In one embodiment, the dilution step maybe performed manually with pipettes and a mixing tube or beaker, andoutside of the analyzer, in which case the dilution ratio will be known.A known volume of the mixture may be deposited in a sample cup 701 FIG.12 on a test cartridge 703 having a serpentine imaging chamber 709. Thesample cup may be in fluid communication to the imaging chamber 709 by achannel 704 at one end of the imaging chamber 709. At the opposite endof the serpentine chamber 709 is a vent hole 707. When the testcartridge is inserted into the analyzer, the vent hole 707 interfaceswith the vacuum/pressure source of the analyzer. The diluted sample isdrawn into and positioned within the imaging chamber 709, when a vacuumis applied at the vent hole 707, such that its entire volume will belocated within the imaging chamber. The diluted sample may also bepushed back into the sample cup by pressure applied at the vent hole andthen pulled back into and positioned in the imaging chamber for thepurposes of mixing the diluted sample, similar to the way describedabove with reference to the mixing chamber 405 in FIG. 8 e . The samplecup may be of the same shape and format as the mixing chamber 405 inFIG. 8 e to facilitate mixing if this is required. For instance, if theknown volume of diluted sample is deposited in the sample cup and thetest cartridge is not inserted in the analyzer for cell analysisimmediately, the cells in the diluted sample may settle to the bottom ofthe sample cup. In this case, mixing may be necessary. Once the dilutedsampled is positioned in the imaging chamber, the analyzer may perform aCBC on the diluted sample as described above.

Alternatively, a measured volume of sample and a measured volume ofdiluent/reagent may be mixed manually, and a portion of the mixturehaving a known volume may be inserted, at a controlled flow rate toprevent crowding and insure a uniform distribution of cells, into a testcartridge having a serpentine imaging chamber as illustrated in FIG. 13. The dimensions of the serpentine path are chosen in accordance withthe dilution ratio, the known volume of the mixture, and the guidelinesset forth above. Every cell in the known volume of the mixture may becounted and analyzed as set forth above.

In another embodiment, the dilution and sample preparation step and maybe performed by an analyzer utilizing a probe for aspirating a wholeblood sample, a shear or face valve for isolating a predetermined volumeof sample, a supply of diluent/stain, a syringe pump for metering anddispensing a known amount of diluent/stain in a mixing bowl for mixingthe sample and diluent/stain, solenoid rocker valves or pinch valves forcontrolling the movements of fluids, vacuum and pressure sources, and adisposable single use test cartridge. The shear valve is in fluidiccommunication with the analyzer probe that aspirates blood samples. Asample is drawn through the probe and into the shear valve, which may beturned, trapping a predetermined amount of sample. The shear valve isfurther turned to a position where it is in fluidic communication with apressure source and the mixing bowl. The isolated blood sample is pushedinto the mixing bowl by the pressure source. The syringe pump is influidic communication with diluent/stain supply and the mixing bowl. Thesyringe pump dispenses a predetermined amount of diluent/stain into themixing bowl. The blood sample and diluent/stain can be mixed in the bowlby pushing air through the probe and bubbling the air through themixture. When mixed, a portion of the mixture may be drawn into ametering chamber of known volume and in fluidic communication with themixing bowl. Optical edge detecting sensors are used to control the flowof the mixture into the metering chamber. The test cartridge illustratedin FIG. 13 is used as a single use disposable cartridge with thehematology analyzer. It is inserted into the analyzer such that thechannel 950 FIG. 13 at one end of the serpentine imaging chamber 951interfaces with, and is in fluidic communication with, the meteringchamber. A vacuum is applied to the opposite end of the serpentine paththrough vent hole 953, FIG. 13 , and the portion of the diluted samplein the metering chamber is drawn into, and positioned in, the imagingchamber, at a controlled rate to prevent crowding and to insure uniformdistribution of the cells, for CBC analysis as described above. Thedimensions of the serpentine path will depend upon the dilution ratio,the volume of the portion of the mixture and the guidelines noted above.One drawback to this arrangement is that the shear valve, meteringchamber, mixing bowl, metering chamber, and connecting fluid channelsmust be flushed between every sample. Such analyzers also requirefrequent calibration and maintenance.

In another embodiment, the dilution step and metering step may beperformed with a test cartridge having a mixing bowl and diluted samplecup on the test cartridge. The analyzer includes a sampling probe,diluent/stain reservoir, precise diluter syringe pump, and wash station.In this case, the probe may be attached to a transfer arm mounted on thebase of cell analyzer 200 FIG. 3 , which can move vertically in the zdirection with respect to stage 225 FIG. 3 . The transfer arm can movehorizontally along a linear axis such that it can be vertically alignedwith the wash station or the diluent/reagent reservoir, or samplecontainer. A schematic of the analyzer is illustrated in FIG. 14 . Thedilution step is as follows. The probe 801 FIG. 14 moves along itslinear axis 803 until it is aligned vertically with the diluent/stainreservoir 805. It then moves downward in the vertical direction untilthe tip of the probe is submerged in the diluent/stain 805. It thenaspirates a known amount of diluent/stain, e.g. 1000 uL. The probesmoves upward and horizontally along axis 803 until it is aligned withthe sample container 807. After aspirating 3 uL of air, it movesdownward in the vertical direction until the tip of the probe is wellsubmerged in the sample in sample container 807. The analyzer thenaspirates 20 uL of sample, such as whole blood, from the samplecontainer, after which the probe is moved upward and above the samplecontainer, where it aspirates another 3 uL slug of air. The probe thenmoves along the linear axis 803 until it is aligned vertically with themixing bowl 721 FIG. 15 on test cartridge 723. The probe is then loweredvertically until the tip of the probe is just above the bottom of themixing bowl 721. The analyzer then dispenses the blood sample and thediluent/stain into the mixing bowl 721 on the test cartridge 723. Thissequence of steps insures that the entire aspirated blood sample isflushed out of the probe by the diluent/stain reagent and into themixing bowl. The analyzer may mix the blood and diluent mechanicallysuch as by moving back and forth along its axis 803 or by bubbling airthrough the mixture, or by aspirating the mixture and redispensing itinto the mixing bowl, or by other methods. After the sample anddiluent/stain are mixed, the analyzer aspirates 20 uL of the mixture.This corresponds to 0.4 uL of undiluted sample. The probe is then movedupwards and moved along the linear axis 803 FIG. 14 until the probe isaligned with the diluted sample reservoir 728 FIG. 15 . The probe islowered and the 20 uL mixture of sample and diluent/stain is dispensedinto sample cup 728 FIG. 15 , which is in fluidic communication with oneend of the serpentine path 719 through channel 731. The mixture may bepulled through the serpentine path of imaging chamber 719 at acontrolled rate by a vacuum source on the analyzer, which interfaceswith vent hole 717 on the opposite end of the imaging chamber from thesample cup 728. Alternatively, after the sample is dispensed, the probemay be positioned into vent hole 717, which may also contain a sealingo-ring, and the analyzer aspirates air through the probe at a controlledrate, such as 2 uL per second, to prevent crowding and to insure auniform distribution of cells. The analyzer positions the entire mixtureof sample and diluent/stain in the imaging chamber 719. The dimensionsof the serpentine path depend upon the dilution ratio, the volume of themixture, and the considerations noted above. Another variation would beto utilize a test cartridge without the sample cup, in which case theprobe, after it has aspirated the 20 uL of diluted sample, may interfacedirectly with the fluidic channel 731 and dispense the mixture at acontrolled rate directly into the serpentine path of imaging chamber719.

The probe may be washed at the wash station 809 FIG. 14 after aspiratingor dispensing blood or diluent/stain or the mixture of sample anddiluent/stain to eliminate any solution adhering to the side of theprobe. Although this process is not described above, those skilled inthe art relating to chemistry analyzers or X-Y dispensing fluidmechanisms will understand the practices and procedures for doing so.

One advantage to this embodiment is the elimination of the shear valveand fluidic tubing and flushing them as well as interconnecting fluidicchannels from sample to sample. It also reduces and may eliminate theneed for pinch and/or solenoid valves.

The analyzer may process samples in parallel by performing the CBCimaging analysis on the known diluted sample in the imaging chamber ofone test cartridge, while the analyzer is simultaneously dilutinganother sample and depositing it in the imaging chamber of a second testcartridge. This can be done to increase analyzer throughput.

EXAMPLES Example 1: Information from Bright-Field and Fluorescent Optics

FIG. 16 shows images that were collected using test devices according tothe present invention. A fluorescent stain Acridine Orange (AO) was usedto differentially stain DNA and RNA of cells in a whole blood sample.The visual images of FIG. 16 were obtained using an Olympus 20X×0.4 NAobjective lens 265 and a Basler 5 MP digital camera 280. Excitation ofthe bright-field images in the second column was provided by white lightbright-field source 260. Excitation of the fluorescent images in thethird column was a 455 nm blue fluorescent light source 270.

White blood cells have significant RNA and DNA and therefore can be seenin the fluorescent images having green and orange structures. The sizeand shape of the green nuclear structure and overall size of the whitecells can be used to differentiate them into sub-groups identified byname in the first column. Notably the basophil and eosinophil sub-groupsof white cells have characteristic features in the bright-field imagedue to the presence of large granules in the cytoplasm. Thereforeembodiments of the present invention make use of both bright-field andfluorescent image analysis to differentiate sub-groups of white cells.

Platelets also take up the AO stain but the size of a platelet issignificantly smaller than any white cell and can therefore bedifferentiated. Because red cells lose their nucleus as they mature,they do not have nuclear material to take up the AO stain. Consequentlythe red cells can be identified as the objects that appear in thebright-field and cannot be seen in the fluorescent field. The immaturered cells, called reticulocytes and the nucleated red blood cells (nRBC)have attributes of red cells but also show small levels of fluorescence.Embodiments of the present invention make use of these combinedattributes to identify and sub-group red blood cells.

Example 2. Statistical Sampling of the Imaging Chamber

Table 1 illustrates a comparison of CBC parameters obtained according tothe present invention and from an automated hematology analyzer.

TABLE 1 Column1 # pairs RBCs WBCs ROI RBC/f RBC/f(%)− WBC/f WBC/f(%)RBC/WBC 100% 9916 2455492 5125 3818.7 643.02 100.0 1.342 100.0 479.12 50% 4958 1229669 2535 1913.7 642.56 99.9 1.325 98.7 485.08  25% 2479623048 1285 968.5 643.28 100.0 1.327 98.9 484.86  10% 992 242197 519373.5 648.48 100.8 1.390 103.5 466.66   5% 496 126186 262 197.2 639.8299.5 1.328 99.0 481.63   1% 100 23683 63 35.6 664.61 103.4 1.768 131.7375.92

-   -   Sample: Low WBC count—approximately 2000/uL (normal is        3,000-10,000/uL).    -   Magnification: 20×    -   Number of images: approximately 10,000 bright-field and 10,000        fluorescent    -   Variable: Column 1— Percentage of total cells use in the        calculation    -   Column #pairs—the number of pairs of images (bright-field plus        fluorescent)    -   Column RBCs— total number of Red Blood Cells counted    -   Column WBCs— total number of White Blood Cells counted    -   Column ROI— total Region of Interest. This is the ‘effective’        number of image frames occupied by actual sample. A frame        totally filled with sample/cells is “1”. A partial frame (due to        an edge or the curved ends of the serpentine shape), is a        fraction of a frame (e.g. 0.567).    -   Column RBC/f—Average number of Red Blood Cells per frame (Column        RBCs divided by Column 5 ROI).    -   Column RBC/f(%)— This is the RBC/frame value at a particular        sampling percentage divided by the RBC/frame for the 100%        sampling case (top line). This is an estimate of the accuracy of        the particular sampling percentage compared to counting 100% of        the cells.    -   Column WBC/f—Average number of White Blood Cells per frame        (Column WBCs divided by Column ROI).    -   Column 9 WBC/f(%)— This is similar to Column 7 but estimates the        accuracy of the sampling percentage for the White Blood Cells.    -   Column RBC/WBC— This is the ratio of RBC/WBC for the particular        sampling percentage.    -   Results: A small percentage of the total frames can provide        accurate results. As a smaller fraction of the total frames are        counted, the accuracy is maintained down to 1% for Red Blood        Cells and down to 5% for White Blood Cells.    -   Discussion: In these experiments, it took approximately one        second to capture an image pair. For this experiment, where        almost 10,000 image pairs were needed to capture 100% of the        sample, this means that image analysis took 10,000 seconds or        approximately 2.8 hours. The experiment shows that the        uniformity of the distribution of cells across the imaging        chamber was good enough to provide accurate results by counting        cells in only 5% of the frames. The goal of “counting every        cell” is achieved because the entire sample size (the Region of        Interest ROI) is measured, but only 5% of the images need to be        analyzed to get accurate results. This reduces the image        analysis time to approximately 8 minutes. It is expected that        advances in camera and computer processing technology will        further reduce this time.

The present invention has now been described in connection with a numberof specific embodiments thereof. However, numerous modifications whichare contemplated as falling within the scope of the present inventionshould now be apparent to those skilled in the art. Therefore, it isintended that the scope of the present invention be limited only by thescope of the claims appended hereto. In addition, the order ofpresentation of the claims should not be construed to limit the scope ofany particular term in the claims.

What is claimed is: 1-69. (canceled)
 70. A method of counting andanalyzing biological particles in whole blood, including cells andplatelets, utilizing an automated microscope, diluent and or staying, asingle use test cartridge having an imaging chamber, the methodcomprising: a) diluting a test sample of whole blood with diluent and/orstain, b) mixing the test sample and diluent and/or stain to asubstantially uniform mixture, c) introducing a portion of the mixtureinto the imaging chamber of a test cartridge, d) introducing the testcartridge with the mixture into an automated microscope, e) capturingone or more digital images of the mixture in the imaging chamber thatare selected to be statistically representative of a number anddistribution of the biological particles in the mixture in the imagingchamber, and f) deriving a quantitative characterization of at least oneaspect of the distribution of the particles in the imaging chamber basedon the images captured.
 71. A method of claim 70 wherein the step ofdriving a quantitative characterization includes analyzing thebiological particles in the captured images with patent recognitionsoftware.
 72. A method of claim 70 wherein the step of deriving aquantitative characterization includes counting all of at least one typeof biological particle in the images using imaging processing software,and calculating the number of particles per unit volume of the one typeof biological particles in the sample.
 73. A method of claim 70 whereinthe capturing of digital images includes brightfield and fluorescentimages.
 74. A method of claim 70 further including the step of waitingfor the particles in the mixture to settle to a bottom of the imagingchamber after the step of transferring the mixture into the imagingchamber.
 75. A method of claim 70 wherein a geometry of the imagingchamber has dimensions that are sufficient that the cells do notoverlap, crowd, or screen when the particles settle to the bottom of theimaging chamber.
 76. A method of claim 70 wherein the step of capturingthe digital images includes capturing images that include all theparticles in the imaging chamber.
 77. A method of claim 70 wherein theamount of sample is 1 μL to 50 μl in the amount of diluent is 10 μl to500 μl.
 78. A method of claim 70 wherein the ratio of diluent to samplein the mixture of diluent and sample is between 10:1 to 250:1.
 79. Amethod of claim 70 wherein the rate of transferring of the mixture ofdiluent and sample is such that the mixture remains substantiallyuniform.
 80. A method of claim 70 wherein the geometry of the imagingchamber is such that the distribution of particles of the mixtureremains substantially homogeneous when the mixture is transferred to theimaging chamber.
 81. A method of claim 70 wherein the shape of theimaging chamber in planar view is serpentine.
 82. A method of claim 81wherein serpentine imaging chamber includes a plurality of convexcurves, wherein each of the convex curves has an inside turning diameterand an outside turning diameter, and wherein the outside turningdiameter of each of the convex curves is about twice the inside diameterof the convex curves of the serpentine imaging chamber.
 83. A method ofclaim 81 wherein the depth of the imaging chamber is uniform throughoutthe length of the imaging chamber and the depth is between 10 μm and 200μm.
 84. A method of claim 81 wherein the width and depth of the imagingchamber is uniform throughout the length of the imaging chamber and thedepth to width ratio of the imaging chamber is greater than 2 to
 1. 85.A method of claim 70 wherein the amount of test sample of whole blood0.1 μL to 5 μL in the amount of diluent and/or stain is 1 μL to 500 μL.86. A method of claim 70 further comprising performing a three-partdifferential of white cells in the sample.
 87. A method of claim 70further comprising performing a five-part differential of white cells inthe sample.
 88. A method of claim 70 wherein the diluting dilutes thetest sample with the diluent and/or stain to achieve a predetermineddilution ratio.